Antibody (immunoglobulin, or also called Ig) proteins have been utilized as pharmaceutical drugs since long ago because of having the function of capturing and eliminating antigens harmful to organisms. Progress in genetic engineering techniques and cell fusion techniques in recent years made it possible to produce monoclonal antibodies that are more homogeneous and have high antigenicity by molecularly designing antibodies that react with their specific antigens and expressing the antibodies in animal cells. These antibody proteins are secreted into cell culture solutions and as such, can be separated, purified, and collected with relative ease.
In general, antibody proteins utilized in immunoassay or immunoblot analysis can be obtained at sufficient yields and purity from natural biological samples such as serum, ascites, or cell culture solutions by using a method utilized in usual protein purification, that is, an ammonium sulfate precipitation method, ion-exchange chromatography, and so on.
On the other hand, separation and purification using these methods for antibody proteins utilized in pharmaceutical drugs or diagnostic drugs or the like, which require high purity, involve contemplating various separation/extraction conditions and using a large number of other chromatography techniques together therewith and also involve optimizing purification conditions for each antibody protein, resulting in a great deal of time and labor. Thus, in the purification of antibody proteins required to be highly pure, affinity chromatography capable of specifically adsorbing the antibody proteins is generally used for conveniently separating and purifying them from other impurities.
Chromatography using a medium comprising an appropriate resin immobilizing thereon proteins such as protein A, protein G, and protein L is utilized most frequently as affinity chromatography having antibody-binding ability. Among these proteins, particularly the protein A is often utilized as a ligand on a medium for purification. The protein A is one kind of cell wall protein with a reported molecular weight of approximately 42,000 produced by a Gram-positive bacterium Staphylococcus aureus. Its structure is composed of seven functional domains (from the amino terminus, signal sequence S, immunoglobulin-binding domain E, immunoglobulin-binding domain D, immunoglobulin-binding domain A, immunoglobulin-binding domain B, immunoglobulin-binding domain C, and Staphylococcus aureus cell wall-binding domain X) (see Non-Patent Documents 1, 2, and 3). These five immunoglobulin-binding domains (domains E, D, A, B, and C) of the protein A can respectively bind to immunoglobulin through its Fc region (see Non-Patent Document 3).
The relative affinity of this protein A for the immunoglobulin-binding domains has been known to depend on many factors such as pH, the types of Staphylococcus aureus strains (Cheung, A. et al., Infec. Immun. 1987. 55: 843-847), and immunoglobulin class (IgG, IgM, IgA, IgD, and IgE) and subclass (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and these domains particularly show strong binding to the Fc region of human IgG1, IgG2, and IgG4, and mouse IgG2a, IgG2b, and IgG3 among immunoglobulin class. The protein A having these properties can bind to immunoglobulin without impairing antigen-binding ability, affinity, and properties as immunoglobulin, and as such, has been used widely as a ligand on a medium for purification of immunoglobulin, particularly IgG, used in various diagnoses, pharmaceutical drugs, and basic researches.
Alternatively, interest has recently been directed toward its application to such cancer therapy that serum blocking factors (composed of specific antigens, antibodies, anti-globulins, and immune complexes), which inhibit the cytotoxicity of sensitized peripheral blood lymphocytes to tumor cells, are adsorbed to protein A and thereby removed from the serum of a patient with tumor (see Patent Documents 1 to 3). Furthermore, protein A has, in addition to IgG-binding activity, the action of activating polyclonal antibody synthesis and has therefore been expected to be used for not only the initial application as a purification resin ligand but also various applications in biotechnology fields.
In an initial process for producing protein A, its separation and purification have been performed directly from the culture solution of Staphylococcus aureus strains. However, due to the problem on the pathogenicity of this bacterium or the contamination by impurities, the process is now shifting toward a producing process that uses a recombinant DNA technique using Escherichia coli (Patent Documents 1 to 3) or a Gram-positive bacterium Bacillus subtilis (Patent Documents 4 to 5). However, the recombinant protein A productivity of Escherichia coli is extremely low, and proteins expressed are not easy to separate and collect because most of them form inclusion bodies or are intracellularly degraded (Non-Patent Document 4). On the other hand, protein A production using Bacillus subtilis, a Gram-positive bacterium, as with Staphylococcus aureus, has adopted a method wherein protein A is secreted and expressed into a medium by adding the signal sequence of a Bacillus subtilis secreted protein to the N-terminus of protein A. This method, when compared with the production system with Escherichia coli, has been reported to provide easy separation and purification and have high productivity (approximately 47 to 100 mg/L) (Fahnestock, S, R. et al., J. Bacteriol. 1986. 165: 796-804). However, the protein A produced in Bacillus subtilis undergoes degradation by extracellular protease intrinsically carried by Bacillus subtilis. Therefore, attempts have been made to use several kinds of extracellular protease-deficient Bacillus subtilis strains (Non-Patent Document 5) as hosts. However, the inhibition of degradation of protein A has not been achieved yet.    [Patent Document 1] Japanese Patent Application No. 07-187019    [Patent Document 2] U.S. Pat. No. 5,151,350    [Patent Document 3] European Patent No. EP0107509    [Patent Document 4] U.S. Pat. No. 4,617,266    [Patent Document 5] European Patent No. EP0124374    [Non-Patent Document 1] Lofdahl, S et al., Proc. Natl. Acad. Sci. USA. 1983. 80: 697-701.    [Non-Patent Document 2] Shuttleworth, H. L et al., Gene. 1987. 58: 283-295.    [Non-Patent Document 3] Uhlen, M. et al., J. Bio. Chem. 1984. 259: 1695-1702.    [Non-Patent Document 4] Nilsson, B et al., Protein Eng. 1987. 1: 107-113.    [Non-Patent Document 5] Fahnestock, S. R et al., Appl. Environ. Microbiol. 1987. 53: 379-384.    [Non-Patent Document 6] Brigido, M et al., J. Basic Microbiology. 1991. 31: 337-345.    [Non-Patent Document 7] Sjostrom, J, -E et al., J. bacteriol. 1975. 123: 905-915.    [Non-Patent Document 8] Bjorck, L. et al., 1984. J. Immunol. 133, 969-974.    [Non-Patent Document 9] Kastern, W. et al., J Biol. Chem. 1992. 267: 12820-12825    [Non-Patent Document 10] Udaka, S. et al., Method Enzymol. 1993. 217: 23-33.